Research Article Received: 13 November 2013
Revised: 3 February 2014
Accepted: 3 February 2014
Published online in Wiley Online Library
Rapid Commun. Mass Spectrom. 2014, 28, 917–920 (wileyonlinelibrary.com) DOI: 10.1002/rcm.6867
Study on the detection limits of a new argon gas cluster ion beam secondary ion mass spectrometry apparatus using lipid compound samples Makiko Fujii1*, Shunichirou Nakagawa2, Kazuhiro Matsuda3, Naoki Man3, Toshio Seki2,4, Takaaki Aoki4,5 and Jiro Matsuo1,4 1
Quantum Science and Engineering Center, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan Department of Nuclear Engineering, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan 3 Surface Analysis Laboratories, Toray Research Center, Inc., 3-7 Sonoyama 3-chome, Otsu, Shiga 520-8567, Japan 4 CREST, Japan Science and Technology Agency (JST), Chiyoda, Tokyo 102-0075, Japan 5 Department of Electronic Science and Engineering, Kyoto University, Nishikyo-ku, Kyoto 615-8510, Japan 2
RATIONALE: Ar gas cluster ion beam secondary ion mass spectrometry (Ar-GCIB SIMS) has been developed as one of the most powerful tools used for analyzing complex biological materials because of its distinctively high secondary ion yield of large organic molecules. However, for the practical analysis of minor components in complex biological materials, the sensitivity of the technique is still insufficient. METHODS: The detection limits of our original Ar-GCIB SIMS apparatus were investigated by measuring lipid compound samples in positive ion mode. The samples were mixtures of 1,2-distearoyl-sn-glycero-3-phosphocholine (C44H88NO8P, DSPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (C40H80NO8P, DPPC). The primary ions were accelerated with 10 keV and the mean cluster size was 1500. The secondary [M+H]+ ions emitted from the sample were analyzed using an orthogonal acceleration time-of-flight mass spectrometer (oa-TOF-MS). In addition, the isotope abundance ratio and the ratio of the [M+H]+ ion signal to the fragment ion signal acquired with Ar-GCIB SIMS were compared with those obtained with conventional Bi cluster SIMS. RESULTS: Secondary [M+H]+ ions and some characteristic fragment ions were clearly observed with high quantitative accuracy in the mass spectra acquired with Ar-GCIB SIMS. The results were clearly better than those obtained with conventional Bi cluster SIMS. CONCLUSIONS: The detection limit of Ar-GCIB SIMS was found to be below 0.1% and was much lower than that of conventional Bi cluster SIMS because of the high [M+H]+ ion yield and the low background. The results suggested that the new Ar-GCIB SIMS apparatus has the capability to acquire valuable information on complex biological materials. Copyright © 2014 John Wiley & Sons, Ltd.
In recent years, analysis of biological samples has been extensively performed in pharmacokinetic and metabolic studies. Matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry (TOF-MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS) are now widely used in the analysis of such samples.[1–3] On the other hand, the use of secondary ion mass spectrometry (SIMS) with conventional monomer ion beams, such as Ar+, Cs+ or Ga+, in biological applications is difficult because of the low yield of secondary molecular ions and complicated fragment ion signals. The use of cluster ions as primary projectiles in SIMS has spread dramatically in the past decade.[4–7] This development was primarily caused by the observation that intact molecular ions could be sputtered from organic and biological samples even at fluences far above the static limit.[8] In particular, the use of a large Ar gas cluster
Rapid Commun. Mass Spectrom. 2014, 28, 917–920
917
* Correspondence to: M. Fujii, Quantum Science and Engineering Center, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan. E-mail: [email protected]
ion beam (Ar-GCIB) has increased considerably and some of these studies showed that bombardment of organic solids with Ar-GCIB triggers a soft emission process so that large intact molecules or even molecular clusters could be desorbed, while the fraction of fragment ions formed was small.[9,10] This was the rationale behind the development of Ar-GCIB SIMS. However, our knowledge of detection limit, sensitivity and matrix effects is still insufficient for the practical analysis of minor components included in complex biological samples. Biological samples are crude and they may contain numerous types of biomolecules. Lipids were the primarily targeted molecules in this study because of their high abundance and low molecular weight (below 1000 Da). More than 100 lipid molecules are found in animal cells and it is essential to identify each one. Therefore, it is important to detect only secondary molecular ion signals, not fragment ion signals. To determine the detection limit of the new Ar-GCIB SIMS apparatus, the spectra of the lipid compound samples were measured and the results were compared with those obtained by conventional Bi cluster SIMS.
Copyright © 2014 John Wiley & Sons, Ltd.
M. Fujii et al.
EXPERIMENTAL Sample preparation The samples studied were DSPC (1,2-distearoyl-sn-glycero-3phosphocholine, C44H88NO8P) and DPPC (1,2-dipalmitoylsn-glycero-3-phosphocholine, C40H80NO8P), the main components of the phospholipid bilayer, from Avanti Polar Lipids, Inc. (Alabaster, AL, USA). The structures of DSPC and DPPC are almost the same and only the length of the fatty acid carbon chain is different, and they were synthetically pure substances. The compounds were dissolved in trichloromethane at a concentration of 2.0 mg/mL. To examine the detection limits, 10%, 1% and 0.1% DPPC were added to DSPC, and the samples were then spin-coated onto the surface of a clean Si wafer. The thickness of the lipid layer was about 100 nm.
The mass analyzer was originally a part of a commercial system, a JMS-T100LC AccuTOF mass spectrometer, manufactured by JEOL Ltd (Tokyo, Japan). It employs a radio-frequency (rf)-only quadrupole ion guide for the transport of secondary ions to the entrance of the TOF analyzer and enables the efficient transmission of ions in a wide m/z range by focusing them on the optical axis.[12] The oa-TOF mass analyzer incorporated a two-stage acceleration and a single-stage reflectron,[13,14] and it enabled rapid measurements to be performed with a DC beam. In this work, the mean cluster size was 1500 and all measurements were performed with an acceleration energy of 10 keV. All spectra were obtained in positive ion mode. The other experimental conditions were as follows: beam current, 40 pA; primary ion dose, 1.0 E +12 ions/cm2; scanned area, 500 μm × 500 μm; and total accumulation time, 10 s.
Ar-GCIB SIMS measurements The measurements were performed in our original SIMS setup at Kyoto University (Kyoto, Japan). Ar-GCIB was adopted as a primary ion beam and an orthogonal acceleration (oa) TOF-MS instrument was used to detect the ions sputtered from the sample. The technique used in this study for cluster formation and ionization has been described in detail elsewhere.[11] In brief, the supersonic expansion of Ar gas at high pressure through a nozzle forms neutral Ar clusters that are collimated into a beam. The neutral cluster beam enters the ionizing chamber after all residual uncondensed gas is skimmed off. Electron ionization is used to obtain an ionized cluster beam that subsequently enters the target chamber and interacts with the target.
Conventional SIMS measurements Bi cluster SIMS has been widely used in the analysis of organic samples because of its high spatial resolution and rather high secondary molecular ion yield.[15] To compare the detection limits, the same samples as examined by Ar-GCIB SIMS were also measured by conventional Bi cluster SIMS, using a TOF.SIMS 5 instrument (ION-TOF GmbH, Münster, Germany) at the Toray Research Center, Inc., Shiga, Japan. The primary ion species in the Bi cluster SIMS experiments was Bi+3 with an acceleration energy of 30 keV. All spectra were obtained in positive ion mode. The other experimental conditions were as follows: beam current, 0.1 pA; primary ion dose, 2.0 E+12 ions/cm2, and scanned area, 350 μm × 350 μm.
Figure 1. Mass spectra of (a) DSPC and (b) DPPC acquired with Ar-GCIB SIMS.
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Figure 2. Mass spectra of (b) DSPC and (c) DPPC acquired with conventional Bi cluster SIMS.
wileyonlinelibrary.com/journal/rcm
Copyright © 2014 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2014, 28, 917–920
Detection limits of a new argon gas cluster ion beam SIMS apparatus Table 1. Comparison of theoretical and experimental isotope abundance of DSPC acquired with Ar-GCIB SIMS and conventional Bi cluster SIMS Ar-GCIB 91 792 793
Theoretical
Experimental
Bi cluster
Theoretical
Experimental
100 49.7 13.7
100.0 52.2 16.6
791 792 793
100 49.7 13.7
100.0 53.9 24.7
Table 2. Intensity ratios of the [M+H]+ ion to the m/z 184.1 fragment ion (see text for details)
DSPC DPPC
Ar-GCIB SIMS
Bi cluster SIMS
5.83% 9.49%
0.0264% 0.0345%
As for the damage cross section, the relationship between secondary ion yield and primary ion dose was confirmed. The secondary ion yield decreased slightly with Ar-GCIB irradiation. In contrast, in Bi cluster ion beam irradiation, the secondary [M+H]+ ion yield of DSPC decreased to 70% at a fluence of 2.0 E+12 ions/cm2, and that was the reason for the selection of the primary ion fluence.
RESULTS AND DISCUSSION Figures 1(a) and 1(b) show the mass spectra of DSPC and DPPC acquired with Ar-GCIB SIMS. The secondary ion intensities in these mass spectra were normalized to the intensity of the base peak at m/z 184.1, the characteristic fragment ion derived from the phosphatidylcholine (PC) head group (C5H15PNO4). In addition to some characteristic fragment ion signals, the [M+H] ions of DSPC (m/z 790.6) and DPPC (m/z 734.6) were clearly detected. Moreover, the secondary ion peaks of [M+H] containing one or two 13C isotopes could be observed in
the mass spectra of both DSPC and DPPC. The individual isotopomers could also be clearly resolved with our high mass resolution Ar-GCIB SIMS apparatus. The mass spectra of DSPC and DPPC acquired with the conventional Bi cluster SIMS are shown, respectively, in Figs. 2(a) and 2(b). For a direct comparison with Fig. 1, the intensity was normalized using the intensity of the m/z 184.1 ion. Even [M+H]+ signals of low intensity could be detected here; various signals belonging to fragment ions below m/z 100 were also observed. The theoretical isotopic abundance distributions of DSPC were compared with the experimental results acquired with Ar-GCIB SIMS and conventional Bi cluster SIMS. The comparison is shown in Table 1, and there was good agreement between the theoretical and experimental results acquired with Ar-GCIB SIMS, indicating that there were no other compounds with the same m/z value as DSPC. On the other hand, the isotope abundances acquired with conventional Bi cluster SIMS were rather high compared with the theoretical ones, especially at m/z 793, because of the high backgrounds and the weak signals obtained with the Bi cluster SIMS apparatus. Furthermore, the intensity ratios of the [M+H]+ ions of DSPC (m/z 790.6) and DPPC (m/z 734.6) to the fragment ion (m/z 184.1) were calculated using the mass spectra acquired with Ar-GCIB SIMS and Bi cluster SIMS, and are shown in Table 2. The ratio with Ar-GCIB SIMS was about 100 times higher than that obtained with conventional Bi cluster SIMS. The mass spectra in the m/z range 730–740 of lipid compound samples acquired by Ar-GCIB SIMS are shown in Fig. 3(a). All the spectra in Fig. 3(a) were offset by 8 E-3,
Rapid Commun. Mass Spectrom. 2014, 28, 917–920
Copyright © 2014 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/rcm
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Figure 3. Mass spectra of lipid compound samples acquired with (a) Ar-GCIB SIMS and (b) conventional Bi cluster SIMS.
M. Fujii et al. high [M+H]+ ion yield and low background obtained with our Ar-GCIB SIMS apparatus. The results presented here suggest that the Ar-GCIB SIMS apparatus has the capability of acquiring valuable information on complex biological samples.
Acknowledgements This study was supported in part by a Research Fellowship for Young Scientists (25-2001) from the Japan Society for the Promotion of Science (JSPS). Figure 4. Standard curves obtained from Ar-GCIB SIMS data (circles) and Bi cluster SIMS data (squares). 1.0 E-2, and 1.2 E-2, except for the 10% data. The signals at m/z 734.6, corresponding to the [M+H] ion of DPPC, decreased quantitatively with the decrease in DPPC volume ratio in the sample. On the other hand, a few peaks that did not decrease with the DPPC volume ratio were obtained around m/z 732. These signals might have originated from a hydrocarbon that was unintentionally mixed into the sample, because these peaks appeared every 14 m/z units. Nevertheless, the mass resolution of our Ar-GCIB SIMS apparatus was high enough that the acquired [M+H]+ signal of DPPC was not affected by this substance. Figure 3(b) shows the mass spectra of the same samples as in Fig. 3(a), measured with conventional Bi cluster SIMS. With the exception of the 10% sample, all the spectra were offset by 8 E-5, 1.0 E-4, and 1.2 E-4. In the mass spectrum of the 1% DPPC sample, a slight [M+H] + ion signal of DPPC was detected. In more diluted samples, no DPPC signal could be obtained due to high backgrounds and low [M+H]+ ion yields. In addition, in order to determine the detection limits, standard curves were obtained using the [M+H]+ ion intensity of DPPC in the mass spectra shown in Fig. 3. First, the ion counts of m/z 734.4–734.8 were collected as representing the intensity of the [M+H]+ ion of DPPC and these were normalized using the intensity of the base peak at m/z 184.1. Then, the signal intensity of the sample with 0% DPPC was subtracted as background from the other samples. The obtained standard curves of Ar-GCIB SIMS and Bi-cluster SIMS are shown in Fig. 4. These standard curves indicated that the detection limit of the novel Ar-GCIB SIMS apparatus was lower than 0.1% in measuring these kinds of lipids. It was also much lower than that of the conventional Bi cluster SIMS because of its high [M+H]+ ion yields and low backgrounds.
CONCLUSIONS
920
The detection limit of Ar-GCIB SIMS was investigated using lipid compound samples and the results were compared with those from conventional Bi cluster SIMS. The [M+H]+ signals of DPPC, the minor component in the studied samples, were clearly observed in the mass spectrum obtained by Ar-GCIB SIMS even in the sample with only 0.1% DPPC. Overall, the detection limit of the Ar-GCIB SIMS apparatus was lower than 0.1% and it was much lower than that of conventional Bi cluster SIMS. The low detection limit is attributed to the
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REFERENCES [1] S. Khatib-Shahidi, M. Andersson, J. L. Herman, T. A. Gillespie, R. M. Caprioli. Direct molecular analysis of whole-body animal tissue sections by imaging MALDI mass spectrometry. Anal. Chem. 2006, 78, 6448. [2] S. J. Atkinson, P. M. Loadman, C. Sutton, L. H. Patterson, M. R. Clench. Examination of the distribution of the bioreductive drug AQ4N and its active metabolite AQ4 in solid tumours by imaging matrix-assisted laser desorption/ionization mass spectrometry. Rapid Commun. Mass Spectrom. 2007, 21, 1271. [3] C. S. Tamvakopoulos, L. F. Colwell, K. Barakat, J. Fenyk-Melody, P. R. Griffin, R. Nargund, B. Palucki, I. Sebhat, X. Shen, R. A. Stearns. Determination of brain and plasma drug concentrations by liquid chromatography/tandem mass spectrometry. Rapid Commun. Mass Spectrom. 2000, 14, 1729. [4] N. Winograd. The magic of cluster SIMS. Anal. Chem. 2005, 77, 142A. [5] A. Wucher. Molecular secondary ion formation under cluster bombardment: A fundamental review. Appl. Surf. Sci. 2006, 252, 6482. [6] C. M. Mahoney. Cluster secondary ion mass spectrometry of polymers and related materials. Mass Spectrom. Rev. 2010, 29, 247. [7] J. S. Fletcher, N. P. Lockyer, J. C. Vickerman. Developments in molecular SIMS depth profiling and 3D imaging of biological systems using polyatomic primary ion. Mass Spectrom Rev. 2011, 30, 142. [8] T. Miyayama, N. Sanada, S. R. Bryan, J. S. Hammond, M. Suzuki. Removal of Ar+ beam-induced damaged layer from polyimide surface with argon gas cluster ion beam. Surf. Interface Anal. 2010, 42, 1453. [9] H. Gnaser, K. Ichiki, J. Matsuo. Sputtered ion emission under size-selected Ar+n cluster ion bombardment. Surf. Interface Anal. 2013, 45, 138. [10] H. Gnaser, M. Fujii, S. Nakagawa, T. Seki, T. Aoki, J. Matsuo. Peptide dissociation patterns in secondary ion mass spectrometry under large argon cluster ion bombardment. Rapid Commun. Mass Spectrom. 2013, 27, 1490. [11] I. Yamada, J. Matsuo, N. Toyoda, A. Kirkpatrick. Molecular processing by gas cluster ion beams. Mater. Sci. Eng. R. 2001, R34, 231. [12] I. V. Chernushevich, A. V. Loboda, B. A. Thomson. An introduction to quadropole-time-of-flight-mass spectrometry. J. Mass Spectrom. 2001, 36, 849. [13] J. H. J. Dawson, M. Guilhaus. Orthogonal-acceleration timeof-flight mass spectrometer. Rapid Commun. Mass Spectrom. 1989, 3, 155. [14] M. Guilhaus, D. Selby, V. Mlynski. Orthogonal acceleration time-of-flight mass spectrometry. Mass Spectrom. Rev. 2000, 19, 65. [15] F. Kollmer. Cluster primary ion bombardment of organic materials. Appl. Surf. Sci. 2004, 231-232, 153.
Copyright © 2014 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2014, 28, 917–920
Revised: 3 February 2014
Accepted: 3 February 2014
Published online in Wiley Online Library
Rapid Commun. Mass Spectrom. 2014, 28, 917–920 (wileyonlinelibrary.com) DOI: 10.1002/rcm.6867
Study on the detection limits of a new argon gas cluster ion beam secondary ion mass spectrometry apparatus using lipid compound samples Makiko Fujii1*, Shunichirou Nakagawa2, Kazuhiro Matsuda3, Naoki Man3, Toshio Seki2,4, Takaaki Aoki4,5 and Jiro Matsuo1,4 1
Quantum Science and Engineering Center, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan Department of Nuclear Engineering, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan 3 Surface Analysis Laboratories, Toray Research Center, Inc., 3-7 Sonoyama 3-chome, Otsu, Shiga 520-8567, Japan 4 CREST, Japan Science and Technology Agency (JST), Chiyoda, Tokyo 102-0075, Japan 5 Department of Electronic Science and Engineering, Kyoto University, Nishikyo-ku, Kyoto 615-8510, Japan 2
RATIONALE: Ar gas cluster ion beam secondary ion mass spectrometry (Ar-GCIB SIMS) has been developed as one of the most powerful tools used for analyzing complex biological materials because of its distinctively high secondary ion yield of large organic molecules. However, for the practical analysis of minor components in complex biological materials, the sensitivity of the technique is still insufficient. METHODS: The detection limits of our original Ar-GCIB SIMS apparatus were investigated by measuring lipid compound samples in positive ion mode. The samples were mixtures of 1,2-distearoyl-sn-glycero-3-phosphocholine (C44H88NO8P, DSPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (C40H80NO8P, DPPC). The primary ions were accelerated with 10 keV and the mean cluster size was 1500. The secondary [M+H]+ ions emitted from the sample were analyzed using an orthogonal acceleration time-of-flight mass spectrometer (oa-TOF-MS). In addition, the isotope abundance ratio and the ratio of the [M+H]+ ion signal to the fragment ion signal acquired with Ar-GCIB SIMS were compared with those obtained with conventional Bi cluster SIMS. RESULTS: Secondary [M+H]+ ions and some characteristic fragment ions were clearly observed with high quantitative accuracy in the mass spectra acquired with Ar-GCIB SIMS. The results were clearly better than those obtained with conventional Bi cluster SIMS. CONCLUSIONS: The detection limit of Ar-GCIB SIMS was found to be below 0.1% and was much lower than that of conventional Bi cluster SIMS because of the high [M+H]+ ion yield and the low background. The results suggested that the new Ar-GCIB SIMS apparatus has the capability to acquire valuable information on complex biological materials. Copyright © 2014 John Wiley & Sons, Ltd.
In recent years, analysis of biological samples has been extensively performed in pharmacokinetic and metabolic studies. Matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry (TOF-MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS) are now widely used in the analysis of such samples.[1–3] On the other hand, the use of secondary ion mass spectrometry (SIMS) with conventional monomer ion beams, such as Ar+, Cs+ or Ga+, in biological applications is difficult because of the low yield of secondary molecular ions and complicated fragment ion signals. The use of cluster ions as primary projectiles in SIMS has spread dramatically in the past decade.[4–7] This development was primarily caused by the observation that intact molecular ions could be sputtered from organic and biological samples even at fluences far above the static limit.[8] In particular, the use of a large Ar gas cluster
Rapid Commun. Mass Spectrom. 2014, 28, 917–920
917
* Correspondence to: M. Fujii, Quantum Science and Engineering Center, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan. E-mail: [email protected]
ion beam (Ar-GCIB) has increased considerably and some of these studies showed that bombardment of organic solids with Ar-GCIB triggers a soft emission process so that large intact molecules or even molecular clusters could be desorbed, while the fraction of fragment ions formed was small.[9,10] This was the rationale behind the development of Ar-GCIB SIMS. However, our knowledge of detection limit, sensitivity and matrix effects is still insufficient for the practical analysis of minor components included in complex biological samples. Biological samples are crude and they may contain numerous types of biomolecules. Lipids were the primarily targeted molecules in this study because of their high abundance and low molecular weight (below 1000 Da). More than 100 lipid molecules are found in animal cells and it is essential to identify each one. Therefore, it is important to detect only secondary molecular ion signals, not fragment ion signals. To determine the detection limit of the new Ar-GCIB SIMS apparatus, the spectra of the lipid compound samples were measured and the results were compared with those obtained by conventional Bi cluster SIMS.
Copyright © 2014 John Wiley & Sons, Ltd.
M. Fujii et al.
EXPERIMENTAL Sample preparation The samples studied were DSPC (1,2-distearoyl-sn-glycero-3phosphocholine, C44H88NO8P) and DPPC (1,2-dipalmitoylsn-glycero-3-phosphocholine, C40H80NO8P), the main components of the phospholipid bilayer, from Avanti Polar Lipids, Inc. (Alabaster, AL, USA). The structures of DSPC and DPPC are almost the same and only the length of the fatty acid carbon chain is different, and they were synthetically pure substances. The compounds were dissolved in trichloromethane at a concentration of 2.0 mg/mL. To examine the detection limits, 10%, 1% and 0.1% DPPC were added to DSPC, and the samples were then spin-coated onto the surface of a clean Si wafer. The thickness of the lipid layer was about 100 nm.
The mass analyzer was originally a part of a commercial system, a JMS-T100LC AccuTOF mass spectrometer, manufactured by JEOL Ltd (Tokyo, Japan). It employs a radio-frequency (rf)-only quadrupole ion guide for the transport of secondary ions to the entrance of the TOF analyzer and enables the efficient transmission of ions in a wide m/z range by focusing them on the optical axis.[12] The oa-TOF mass analyzer incorporated a two-stage acceleration and a single-stage reflectron,[13,14] and it enabled rapid measurements to be performed with a DC beam. In this work, the mean cluster size was 1500 and all measurements were performed with an acceleration energy of 10 keV. All spectra were obtained in positive ion mode. The other experimental conditions were as follows: beam current, 40 pA; primary ion dose, 1.0 E +12 ions/cm2; scanned area, 500 μm × 500 μm; and total accumulation time, 10 s.
Ar-GCIB SIMS measurements The measurements were performed in our original SIMS setup at Kyoto University (Kyoto, Japan). Ar-GCIB was adopted as a primary ion beam and an orthogonal acceleration (oa) TOF-MS instrument was used to detect the ions sputtered from the sample. The technique used in this study for cluster formation and ionization has been described in detail elsewhere.[11] In brief, the supersonic expansion of Ar gas at high pressure through a nozzle forms neutral Ar clusters that are collimated into a beam. The neutral cluster beam enters the ionizing chamber after all residual uncondensed gas is skimmed off. Electron ionization is used to obtain an ionized cluster beam that subsequently enters the target chamber and interacts with the target.
Conventional SIMS measurements Bi cluster SIMS has been widely used in the analysis of organic samples because of its high spatial resolution and rather high secondary molecular ion yield.[15] To compare the detection limits, the same samples as examined by Ar-GCIB SIMS were also measured by conventional Bi cluster SIMS, using a TOF.SIMS 5 instrument (ION-TOF GmbH, Münster, Germany) at the Toray Research Center, Inc., Shiga, Japan. The primary ion species in the Bi cluster SIMS experiments was Bi+3 with an acceleration energy of 30 keV. All spectra were obtained in positive ion mode. The other experimental conditions were as follows: beam current, 0.1 pA; primary ion dose, 2.0 E+12 ions/cm2, and scanned area, 350 μm × 350 μm.
Figure 1. Mass spectra of (a) DSPC and (b) DPPC acquired with Ar-GCIB SIMS.
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Figure 2. Mass spectra of (b) DSPC and (c) DPPC acquired with conventional Bi cluster SIMS.
wileyonlinelibrary.com/journal/rcm
Copyright © 2014 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2014, 28, 917–920
Detection limits of a new argon gas cluster ion beam SIMS apparatus Table 1. Comparison of theoretical and experimental isotope abundance of DSPC acquired with Ar-GCIB SIMS and conventional Bi cluster SIMS Ar-GCIB 91 792 793
Theoretical
Experimental
Bi cluster
Theoretical
Experimental
100 49.7 13.7
100.0 52.2 16.6
791 792 793
100 49.7 13.7
100.0 53.9 24.7
Table 2. Intensity ratios of the [M+H]+ ion to the m/z 184.1 fragment ion (see text for details)
DSPC DPPC
Ar-GCIB SIMS
Bi cluster SIMS
5.83% 9.49%
0.0264% 0.0345%
As for the damage cross section, the relationship between secondary ion yield and primary ion dose was confirmed. The secondary ion yield decreased slightly with Ar-GCIB irradiation. In contrast, in Bi cluster ion beam irradiation, the secondary [M+H]+ ion yield of DSPC decreased to 70% at a fluence of 2.0 E+12 ions/cm2, and that was the reason for the selection of the primary ion fluence.
RESULTS AND DISCUSSION Figures 1(a) and 1(b) show the mass spectra of DSPC and DPPC acquired with Ar-GCIB SIMS. The secondary ion intensities in these mass spectra were normalized to the intensity of the base peak at m/z 184.1, the characteristic fragment ion derived from the phosphatidylcholine (PC) head group (C5H15PNO4). In addition to some characteristic fragment ion signals, the [M+H] ions of DSPC (m/z 790.6) and DPPC (m/z 734.6) were clearly detected. Moreover, the secondary ion peaks of [M+H] containing one or two 13C isotopes could be observed in
the mass spectra of both DSPC and DPPC. The individual isotopomers could also be clearly resolved with our high mass resolution Ar-GCIB SIMS apparatus. The mass spectra of DSPC and DPPC acquired with the conventional Bi cluster SIMS are shown, respectively, in Figs. 2(a) and 2(b). For a direct comparison with Fig. 1, the intensity was normalized using the intensity of the m/z 184.1 ion. Even [M+H]+ signals of low intensity could be detected here; various signals belonging to fragment ions below m/z 100 were also observed. The theoretical isotopic abundance distributions of DSPC were compared with the experimental results acquired with Ar-GCIB SIMS and conventional Bi cluster SIMS. The comparison is shown in Table 1, and there was good agreement between the theoretical and experimental results acquired with Ar-GCIB SIMS, indicating that there were no other compounds with the same m/z value as DSPC. On the other hand, the isotope abundances acquired with conventional Bi cluster SIMS were rather high compared with the theoretical ones, especially at m/z 793, because of the high backgrounds and the weak signals obtained with the Bi cluster SIMS apparatus. Furthermore, the intensity ratios of the [M+H]+ ions of DSPC (m/z 790.6) and DPPC (m/z 734.6) to the fragment ion (m/z 184.1) were calculated using the mass spectra acquired with Ar-GCIB SIMS and Bi cluster SIMS, and are shown in Table 2. The ratio with Ar-GCIB SIMS was about 100 times higher than that obtained with conventional Bi cluster SIMS. The mass spectra in the m/z range 730–740 of lipid compound samples acquired by Ar-GCIB SIMS are shown in Fig. 3(a). All the spectra in Fig. 3(a) were offset by 8 E-3,
Rapid Commun. Mass Spectrom. 2014, 28, 917–920
Copyright © 2014 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/rcm
919
Figure 3. Mass spectra of lipid compound samples acquired with (a) Ar-GCIB SIMS and (b) conventional Bi cluster SIMS.
M. Fujii et al. high [M+H]+ ion yield and low background obtained with our Ar-GCIB SIMS apparatus. The results presented here suggest that the Ar-GCIB SIMS apparatus has the capability of acquiring valuable information on complex biological samples.
Acknowledgements This study was supported in part by a Research Fellowship for Young Scientists (25-2001) from the Japan Society for the Promotion of Science (JSPS). Figure 4. Standard curves obtained from Ar-GCIB SIMS data (circles) and Bi cluster SIMS data (squares). 1.0 E-2, and 1.2 E-2, except for the 10% data. The signals at m/z 734.6, corresponding to the [M+H] ion of DPPC, decreased quantitatively with the decrease in DPPC volume ratio in the sample. On the other hand, a few peaks that did not decrease with the DPPC volume ratio were obtained around m/z 732. These signals might have originated from a hydrocarbon that was unintentionally mixed into the sample, because these peaks appeared every 14 m/z units. Nevertheless, the mass resolution of our Ar-GCIB SIMS apparatus was high enough that the acquired [M+H]+ signal of DPPC was not affected by this substance. Figure 3(b) shows the mass spectra of the same samples as in Fig. 3(a), measured with conventional Bi cluster SIMS. With the exception of the 10% sample, all the spectra were offset by 8 E-5, 1.0 E-4, and 1.2 E-4. In the mass spectrum of the 1% DPPC sample, a slight [M+H] + ion signal of DPPC was detected. In more diluted samples, no DPPC signal could be obtained due to high backgrounds and low [M+H]+ ion yields. In addition, in order to determine the detection limits, standard curves were obtained using the [M+H]+ ion intensity of DPPC in the mass spectra shown in Fig. 3. First, the ion counts of m/z 734.4–734.8 were collected as representing the intensity of the [M+H]+ ion of DPPC and these were normalized using the intensity of the base peak at m/z 184.1. Then, the signal intensity of the sample with 0% DPPC was subtracted as background from the other samples. The obtained standard curves of Ar-GCIB SIMS and Bi-cluster SIMS are shown in Fig. 4. These standard curves indicated that the detection limit of the novel Ar-GCIB SIMS apparatus was lower than 0.1% in measuring these kinds of lipids. It was also much lower than that of the conventional Bi cluster SIMS because of its high [M+H]+ ion yields and low backgrounds.
CONCLUSIONS
920
The detection limit of Ar-GCIB SIMS was investigated using lipid compound samples and the results were compared with those from conventional Bi cluster SIMS. The [M+H]+ signals of DPPC, the minor component in the studied samples, were clearly observed in the mass spectrum obtained by Ar-GCIB SIMS even in the sample with only 0.1% DPPC. Overall, the detection limit of the Ar-GCIB SIMS apparatus was lower than 0.1% and it was much lower than that of conventional Bi cluster SIMS. The low detection limit is attributed to the
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Rapid Commun. Mass Spectrom. 2014, 28, 917–920
